Phospholipase A2 (PLA2) plays a central role in membrane phospholipid homeostasis, phospholipid remodeling, and general phospholipid metabolism. This enzyme also catalyzes the release of arachidonic acid, the key first step in the biosynthesis of the eicosanoids, including prostaglandins and leukotrienes. PLA2 is also implicated in signal transduction. We have performed extensive studies in the past with the PLA2 from cobra venom (N. naja naja) and have used this enzyme as a model for investigating enzyme-lipid interactions. We now plan to apply these same approaches to studying the newly discovered mammalian cellular PLA2s. This research proposal focuses on characterizing novel cellular PLA2s and also continues our studies on lipid hydrolysis with the cobra venom PLA2. In particular, we will investigate a novel Ca2+-independent PLA2 from the mouse macrophage-like cell line, P388D1. These studies will include kinetic characterization and an investigation into the regulation of this enzyme by ATP and by enzyme oligomerization. We also plan to sequence and clone this enzyme. We will determine the expression levels of three low molecular weight PLA2s in P388D1 cells. Two of these enzymes are novel low molecular weight PLA2s which we will express and characterize, one with 16-Cys residues (Group IIC) and one with 12-Cys residues (Group V). We will continue our investigation of lipolytic enzyme hydrolysis with cobra venom PLA2 and will extend these studies to include various mammalian PLA2s where appropriate. These studies will include investigation of interfacial activation and lipid activation and will help to determine the overall rate-limiting step of PLA2 action. These aims will be accomplished by a combination of kinetic analysis, equilibrium dialysis, heavy atom isotope studies, and site-directed mutagenesis.